American Journal of Life Sciences

2015; 3(2): 85-92

Published online March 3, 2015 (http://www.sciencepublishinggroup.com/j/ajls)
doi: 10.11648/j.ajls.20150302.15
ISSN: 2328-5702 (Print); ISSN: 2328-5737 (Online)

Molecular Mechanism of Formalin-Induced Toxicity and Its

Management
Alpana Khatun1, Md Masud Rana1, Md Rafiqul Islam Khan1, Mir Imam Ibne Wahed1,
Md. Anwar Habib2, Md. Nazim Uddin2, Zakia Sultana Sathi3, A. R. M. Ruhul Amin4,
Abu Syed Md Anisuzzaman1, 4, *
1
Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh
2
Department of Pharmacology and Therapeutics, Rajshahi Medical College, Rajshahi-6000, Bangladesh
3
Department of Pharmacy, Daffodil International University, Dhaka 1207, Bangladesh
4
Department of Hematology & Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA

Email address:
a_zamanpan@yahoo.com (A. S. M. Anisuzzaman), aanisuz@emory.edu (A. S. M. Anisuzzaman)

To cite this article:

Alpana Khatun, Md Masud Rana, Md Rafiqul Islam Khan, Mir Imam Ibne Wahed, Md. Anwar Habib, Md. Nazim Uddin, Zakia Sultana
Sathi, A. R. M. Ruhul Amin, Abu Syed Md Anisuzzaman. Molecular Mechanism of Formalin-Induced Toxicity and Its Management.
American Journal of Life Sciences. Vol. 3, No. 2, 2015, pp. 85-92. doi: 10.11648/j.ajls.20150302.15

Abstract: The use of formalin (40% formaldehyde) for the preservation of food in an illegal way becoming a serious health
issue in developing countries including Bangladesh. We investigated the Formalin (FA)-induced organ toxicity in Swiss albino
mice. FA induction caused the significant elevation of the liver enzyme, SGOT and SGPT; the MDA levels in the liver and
brain. Among the fractions of methanol extract of L. globosus, ethyl acetate (EA) fraction significantly reduced the elevated
biochemical parameters (FA vs FA + EA fraction, Ka/L); SGOT (78.4 0.3 vs 14.3 0.9), SGPT (100.5 5.2 vs 14.6 0.7),
MDA in liver (10.9 0.2 vs 5.6 0.1) and MDA in brain (16.9 0.2 vs 6.3 0.2). Morphological analyses also supported the
beneficial effect of EA fraction in FA-induced liver toxicity. FA induction caused the phosphorylation of JNK, member of
mitogen activated protein kinase (MAPK) in both the liver and brain, which were completely abolished by the treatment of EA
fraction of L. globosus. Chemical analyses showed that the EA fraction exhibited antioxidant and free radical scavenging
properties. The protective effect of the EA fraction on the FA-induced toxicity by the modulation of oxidative inflammatory
pathway by its antioxidant and free radical scavenging activity.
Keywords: Loranthus globosus, SGOT and SGPT, Antioxidant and Free Radical Scavenging, JNK Phosphorylation,
Formalin

1. Introduction
Formaldehyde is one of the common environmental agents
found in tobacco smoke, paint, diesel, gasoline exhaust, and
medical and industrial products (Flyvholm and Andersen
1993). It has been considered to be potentially carcinogenic
that makes it a subject of major environmental concern (Heck,
Casanova et al. 1990). In Bangladesh, FA is used as an illegal
practice to preserve fish, fruits and vegetables which is
dangerously affecting the health of local peoples. FA is an
extremely reactive chemical, and reacts with monoamines or
amides to form methylene bridges and produces covalently
cross-linked complexes with proteins and DNA (Saito,
Nishio et al. 2005). In addition to DNAprotein cross-links, it
has been reported that FA modulates the cellular glutathione
(GSH)status and generates oxidative free radicals (Teng,
Beard et al. 2001, Saito, Nishio et al. 2005). Some studies
have linked chronic FA exposure not only to cancer incidence,
but also to teratogenicity, and to a variety of
neurodegenerative and vascular disorders (Kilburn 1994,
Sakanashi, Rogers et al. 1996, Yu, Wright et al. 2003).
When ingested, FA is rapidly metabolized and removed
from the liver. The major metabolic enzymes, particularly
NAD-dependent aldehyde dehydrogenase and GSH-
dependent formaldehyde hydrogenase are involved in the
metabolism of FA, which has been detected in human liver
and red blood cells and in a number of animal tissues, such as
respiratory and olfactory epithelium in the rat (Teng, Beard et
 American Journal of Life Sciences 2015; 3(2): 85-92
al. 2001, Bakand, Hayes et al. 2005).
Various exogenous agents including FA can induce the
generation of reactive oxygen species (ROS)(Riley 1994). An
excess generation of ROS in cells is known to damage DNA,
lipids and proteins, resulting in a number of untoward
pathophysiological effects such as mutagenesis, malignant
transformation, cell death etc.(Wiseman and Halliwell 1996,
Berlett and Stadtman 1997, Thannickal and Fanburg 2000).
At the initial stage, to eliminate deleterious ROS from the
body, cells utilize various enzymatic and non-enzymatic
antioxidants. However, due to excessive oxidative stress, the
bodys endogenous antioxidant source become exhausted
which necessitates the supply of exogenous antioxidants.
In the present study, we investigated the antioxidant
compound(s) in the methanolic extracts and its various
fractions of Loranthus globosus and evaluated their
protective effects against FA-induced organ injury in mouse
model. We have analyzed serum glutamate oxaloacetate
transaminase (SGOT) and serum glutamate pyruvate
transaminase (SGPT) levels as biomarkers for liver injury,
malondialdehyde (MDA) as biomarker for lipoid
peroxidation in liver and brain. The signaling mechanisms of
FA-induced oxidative damage and the protective effects of L.
globosus were also investigated.
2. Materials and Methods
2.1. Reagents
Dexamethasone was gifted from the Chemico
pharmaceuticals Ltd., Rajshahi, Bangladesh. 0.9% NaCl
solution (Beximco Infusion Lab., Dhaka, Bangladesh),
SGOT and SGPT (AMP Medizintechnik GmbH; Austria),
gallic acid standard, trichloro acetic acid, thiobarbituric acid,
MDA standard and n-butanol (GE Health care,
Buckinghamshire, UK) , -diphenyl--picrylhydrazyl
(DPPH), sodium phosphate, ammonium molybdate, ascorbic
acid and methanol (Sigma Aldrich, St. Louis, USA), anti-c-
Jun N-terminal kinase (JNK), anti-phospho-JNK and anti-
actin (Cell Signaling Technology, Inc., Massachusetts, USA)
were obtained from the sources noted. All employed
chemicals and solvents were of analytical grade.
2.2. Plant Materials
The barks of L. globosus were collected from Pabna,
Bangladesh and were taxonomically identified by Dr. Md.
Anisuzzaman, Associate Professor, Department of Botany,
University of Rajshahi, Bangladesh and the voucher
specimen has been preserved there. The cleaned stem barks
were dried under sunshine and subsequently in oven at 50C
temperatures for complete dryness.
2.3. Extraction and Fractionation of Plant Materials
The dried and pulverized plant material was cold extracted
by methanol as described previously (Khan, Islam et al. 2010)
and the methanolic extract (ME) was successively partitioned
with petroleum ether (PE), chloroform (CF) and ethyl acetate
86
(EA) using modified Kupchan partitioning method (BC, R et
al. 1993). The resultant fractions were then evaporated by
roto-dryer at low temperature (40-50C) to dryness. The
fractions were preserved at -20C until use. Dimethyl
sulfoxide was used as a solvent for the preparation of dose of
various fractions.
2.4. Chemical Analysis of the Methanol Extract and Its
Fractions
2.4.1. Determination of Total Phenolic (TP) Content
The total phenolic (TP) content of the crude methanolic
extract (ME) of L. globosus and its various fractions (PE, CF,
EA) were determined by Folin-Ciocalteu Reagent (FCR)
according to the method of Kumar et al. (Kumar, Ganesan et
al. 2007) with slight modification. Briefly, the solution of
each extract (0.5 ml, 1mg/ml) was diluted to 10 ml with
distilled water in a volumetric flask. FCR (1 ml) was added
and mixed thoroughly, and then sodium carbonate solution (3
ml, 2%) was added. After 2h incubation at room temperature,
absorbance was measured at 760 nm. The total phenolic
content was determined by comparison with the standard
calibration curve of gallic acid, and results are presented as
mg of gallic acid equivalents (mg of GAE) per gram dry
weight of extracts. All tests were conducted in triplicate.
2.4.2. Determination of Total Flavonoid (TF) Content
The total flavonoid content of each extract was estimated
by Zhishen et al.(Zhishen, Mengcheng et al. 1999). Briefly,
0.5 ml (1 mg/ml) of each sample was mixed with 2 ml of
distilled water and subsequently with 0.15 ml of NaNO2
solution (15%). After incubation for 6 min, 0.15 ml of AlCl3
solution (10%) was added and allowed to stand for another 6
min. Then 2 ml of NaOH solution (4%) was added to the
mixture and adjusted the final volume to 5 ml by distilled
water. The mixture was then mixed thoroughly and allowed
to stand for another 15 min. The absorbance of the final
solution was determined at 510 nm. The total flavonoid
content was determined by comparison with the standard
calibration curve of gallic acid, and results are presented as
mg of gallic acid equivalents (mg of GAE) per gram dry
weight of extracts.
2.5. In Vitro Antioxidant Assay
2.5.1. DPPH Radical Scavenging Assay
The plant extracts were tested for the scavenging effect on
DPPH radical according to the method of Pan et al. (Pan,
Wang et al. 2008). Accordingly, 0.2 ml of extract solution in
ethanol (95%) at different concentrations (1, 2, 4, 8, 16, 32
and 64 g/ml, respectively) was added to 8 ml of 0.004%
(w/v) stock solution of DPPH in ethanol (95%). The
scavenging activity on the DPPH radical was determined by
measuring the absorbance at 517 nm until the reaction
reached the steady state, using a UV-visible
spectrophotometer (Shimadzu, Tokyo, Japan). Ascorbic acid
was used as a positive control. The DPPH radical scavenging
activity (S%) was calculated using the following formula:
87 Alpana Khatun et al.: Molecular Mechanism of Formalin-Induced Toxicity and Its Management
S% = [(Acontrol Asample)/A control]100
2.5.2. Total Antioxidant Activity Assay
The total antioxidant activity of the methanolic extract and
its various fractions of L. globosus were assessed by
phosphomolybdenum method as described previously (Prieto,
Pineda et al. 1999). Briefly, 0.5 ml sample solution of each
fraction was mixed with 3 ml of phosphomolybdenum
solution comprising: 0.6 M sulphuric acid, 28 mM sodium
phosphate and 4 mM ammonium molybdate. The mixture
was then incubated at 95C for 90 min followed by cooling at
room temperature. The absorbance of the solution was
measured at 695 nm against blank. Ascorbic acid was used as
a positive control.
2.6. Induction of FA-Induced Organ Injury in Mouse
Model
Male Swiss albino mice weighing about 25 - 30gm were
used for the FA-induced animal model study. To measure FA-
induced toxicity, we adopted several routes of administration
as follows:
i. Oral administration of 4% FA mixing with fruits daily
for one month.
ii. Oral administration of 4% FA with drinking water daily
for one month.
iii. Intraperitoneal (IP) administration of 4% FA (0.1ml/day)
for seven days.
iv. Subcutaneous (SC) administration of 4% FA (0.1ml/day)
through hind paws for seven days.
2.7. Analysis of Various Biochemical Parameters
After completion of treatment (FA, drug, plant extract),
mice were sacrificed and approximately 2-3 ml of blood was
collected directly from heart by syringes, centrifuged at
4000rpm for 30minutes to collect supernatants. Serum GOT
and GPT levels were measured by UV-visible
spectrophotometric method using commercial wet reagent
diagnostic kits (AMP Medizintechnik GmbH; Austria)
according to the manufacturers protocol. Briefly, the amount
of oxaloacetate and pyruvate formed by each of the two
assays were measured by means of the 2, 4 -
dinitrophenylhydrazone of pyruvic acid, the color of which
was read at 520 nm by spectrophotometer. The intensity of
color was proportional to the amount of enzyme in each
sample.
2.8. Analysis of Malondialdehyde (MDA)
The MDA levels on the tissues were determined by the
method of Draper and Hadley based on the reaction of MDA
with thiobarbituric acid (TBA) at 95C (Draper and Hadley
1990). The brain and liver tissues were homogenized
separately on ice in MDA lysis buffer and centrifuged at
13,000g for 10 min to collect the supernatant. In the TBA
test reaction, MDA and TBA react to form a pink pigment
with absorption maximum at 532 nm. The reaction was
performed at pH 2-3 at 95C for 15 min. The supernatant was
mixed with 2.5volumes of 10% (w/v) trichloroacetic acid to
precipitate the protein. The precipitate was pelleted by
centrifugation and supernatant was reacted with 0.67% TBA
in a boiling water bath for 15 min. After cooling, the
absorbance was read at 532nm. Arbitrary values obtained
were compared with a series of standard solutions (1, 1, 3, 3-
tetramethoxypropane). Results are expressed as nmol per
milligram of tissue.
2.9. Immunoblotting
The liver and brain samples from the respective animal
groups were dissected and placed immediately into ice-cold
phosphate-buffered saline (PBS). The collected tissues were
homogenized by sonication with an ultrasonic homogenizer
(VP-050, Taitec Corp., Koshigaya, Japan) followed by lysis
with RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.25% Na-
deoxycholate, and 150mM NaCl, 1 mM Na3VO4 and NaF)
containing protease inhibitors (1 g/ml each of EDTA and
phenylmethylsulfonyl fluoride). Cell lysates were centrifuged
at 12,000g for 15 min at 4C. The supernatant was collected
and boiled with SDS sample buffer (12 mM Tris-HCl, 10%
glycerol, 10% sodium dodecyl sulfate and 1% 2-
mercaptoethanol and 0.1% bromophenol blue, pH 6.8) for 5
min at 100C. Proteins were separated by SDS-PAGE and
transferred to polyvinylidene fluoride (PVDF) membranes.
Membranes were probed with appropriate concentrations of
primary antibody. The immunoreactive proteins were
detected by horseradish-peroxidase-labeled secondary
antibody with Amersham ECL advance Western blotting
Detection Kit (GE healthcare).
2.10. Histopathological Study
After seven days of observation, the animals were killed
by cervical dislocation. The liver was carefully excised,
rinsed in cold sucrose solution, and blotted in dry filter paper.
The specimen was fixed with 10% buffered-formalin and
dehydrated in ascending order of ethanol and embedded in
paraffin. The blocks were sectioned with the help of rotation
microtome at 6-micron thickness. The sections were
subjected to Hematoxylin and Eosin staining procedures and
the histological examination was done with the aid of the
high power microscope. The histological outline of each
photomicrograph was conducted through a stereological grid
in order to access the population of the cells in each organ.
The permanent photomicrographs of each slide were
recorded with a Kodak Digital Camera for subsequent
histological analysis.
2.11. Statistical Analysis
Data were expressed as mean standard error of mean
(SEM). Statistical comparisons were performed by one-way
analysis of variance (ANOVA), followed by Scheffes post-
hoc test or students paired or unpaired t-test where
appropriate. Results were considered to be significant when p
values were less than 0.05 (p<0.05). Statistical calculations
and the graphs were prepared using Graph Pad Prism version
 American Journal of Life Sciences 2015; 3(2): 85-92 88

5.00 for Windows (Graph Pad Software, San Diego, CA, flavonoid (TF) compounds in the various fractions (PE, CF
USA, www.graphpad.com). and EA) of methanolic extracts of L. globosus. The results
are shown in Table 1. The highest amount of TP and TF
3. Results contents were found in EA fractions (114.84 0.20 and
276.90 16.34 GAE per gram, respectively).
3.1. Chemical Analysis of Total Phenol and Flavonoid
Contents of the Crude Methanol Extract (ME) and Its 3.2. In Vitro Antioxidant Activity Analysis of the Crude
Fractions of L. globosus Methanol Extract (ME) and Its Fractions of L.
globosus
Table 1. Determination of total phenol and flavonoid contents in the crude
methanol (ME) extract and its various fractions of L. globosus. The Fig. 1A shows that there was significant scavenging of
DPPH free radicals on various fractions of L. globosus.
Total phenol Total flavonoid
Sample Maximum scavenging of 95.6 2.5% was observed by EA
GAE/gm of dried extracts GAE/gm of dried extracts
ME extracts 73.07 0.08 198.43 5.19 fractions in a similar extent to that of standard ascorbic acid
PE fractions 14.03 0.03 102.50 11.3 (94.3 4.2%), followed by CF (62.0 1.2%) and PEF (61.5
EA fractions 114.84 0.20 276.90 16.34 2.4%), respectively.
CF fractions 31.84 0.04 53.21 6.09 Similarly, the Fig. 1B shows that the EA fractions have the
highest total antioxidant activity. The order of total
The phenolic and flavonoid compounds are considered as
antioxidant activity in various fractions of L. globosus were
potential antioxidants and free radical scavengers. Here, we
EA>PE>CF. The results are concordant with the contents of
investigated the contents of total phenol (TP) and total
total phenolic and total flavanoid in various fractions.

Fig. 1. In vitro antioxidant activity of the methanol extract and fractions of L. globosus: (A) DPPH radical scavenging activity of crude methanol extracts (ME)
and its various fraction of L. globosus. (B) Total antioxidant activity of the crude methanol extract (ME) and its various fractions of L. globosus. Ascorbic acid
was used as standard scavenger of the oxidant as well as standard antioxidant agent.

3.3. Time Course of FA-Induced Liver Injury

Fig. 2. Time course of FA-induced elevation of liver enzymes (SGOT and SGPT) in various routes of administration. FA was supplied by (A) intraperitoneal
injection; (B) hind paw injection; (C) oral route with drinking water and (D) oral route with fruits. indicates significantly different (p<0.05) from time
control after treatment with FA.
89 Alpana Khatun et al.: Molecular Mechanism of Formalin-Induced Toxicity and Its Management

In order to estimate the FA-induced liver injury, we

measured SGPT and SGOT levels at various time periods
upon FA treatment for different routes of administration. The
intraparetoneal (IP) route of administration shows
approximately 10-fold maximum increase of both the liver
enzymes (Fig. 2A) on 6th day of administration as compared
to control. In the case of oral routes, similar increase of liver
enzymes were achieved at longer duration (15-30 days) as
shown in Fig. 2C and 2D. Elevated level of both the enzymes
was least when injected in the hind paw (Fig. 2B). Because
of the difficulty in dose adjustment along with administration Fig. 3. Dose dependent effect of ethyl acetate froction of L. globosus on
in oral route and less sensitivity to hind paw, we chose IP formalin induced elevated SGOT and SGPT. Reduction of FA-induced
administration for 7-day FA treatment period in our elevated SGOT and SGPT by EA fraction of L. globosus in a dose-dependent
experimental model. manner.* indicates significant (p<0.05) elevation of SGOT and SGPT after
FA induction as compared to normal control. # indicates significant (p<0.05)
3.4. Ethyl Acetate (EA) Fraction of L. globosus Reduces the reduction of SGOT and SGPT upon treatment of EA fraction as compared to
FA-Induced Elevated SGOT and SGPT Levels in a FA-induced control.
Dose-Dependent Manner
3.5. Ethyl Acetate (EA) Fraction of L. globosus Suppress
Since the EA fraction of L. globosus possesses the the FA-Induced Lipid Peroxidation
maximum amount of antioxidant compounds (TP and TF),
Malondialdehyde (MDA) is one of the most important
we investigated the effect of this fraction on the FA-induced
biomarker of lipid peroxidation, which is generated due to
elevated SGOT and SGPT levels. Single IP injection of EA
the excessive load of free radicals. In the present study, we
fraction reduced the FA-induced elevation of SGOT and
found that IP administration of FA caused the elevation of
SGPT levels in a dose dependent manner (Fig. 3). EA
serum and brain MDA levels which were suppressed
fraction at 12-mg/kg-body weight reduced the elevated
significantly by the co-administration of single IP injection of
SGOT and SGPT levels completely. 12-mg/kg-body weight
EA fraction of L. globosus in a similar extent to that of
of EA fraction of L. globosus were used for further
standard anti-inflammatory drug, dexamethasone. The results
experiments unless mentioned.
are shown in Fig. 4A and 4B.

Fig. 4. Effects of EA fraction of L. globosus on FA-induced elevated MDA level. (A) FA-induced elevated liver MDA level was significantly reduced by the EA
fraction of L. globosus in a similar extent to that of anti-inflammatory drug, dexamethasone. (B) FA-induced elevated brain MDA level was significantly
reduced by the EA fraction of L. globosus in a similar extent to that of anti-inflammatory compound, dexamethasone (Dexa). * indicates significant (p<0.05)
elevation of MDA level after FA induction as compared to normal control. # indicates significant (p<0.05) reduction of MDA level after treatment of EA
fraction as compared to FA-induced control. Concentration of dexamethasone and EA fraction of L. globosus were 2 and 12 mg/kg body weight,
respectively.Values are mean SEM (n = 4).

3.6. Histopathology of Liver Tissues after FA-Induced
Damage
Liver tissues were collected from the control and treatment
group of mice were fixed and stained with Hematoxylin and
Eosin as described in methods. The specimens were then
visualized under microscope with 40 magnifications. As
shown in Fig. 5B, there was swelling of liver tissues with
marked fatty degeneration with FA treatment. Areas of focal
necrosis were also observed in the liver of the FA-induced
mice as compared to the control (Fig.5A). The
histopathological degeneration was repaired considerably by
the supplementation of EA fraction of L. globosus as shown
in Fig. 5D.
 American Journal of Life Sciences 2015; 3(2): 85-92 90

Fig. 5. Histopathological studies of FA-induced liver damage with or without the treatment of EA fraction of L. globosus and Dexamethasone. The
representative photographs shown are (A) normal control (B) FA-induced control (C) FA-induced mice with the supplementation of anti-inflammatory
compound, dexamethasone and (D) FA-induced mice with the supplementation of EA fraction of L. globosus. Scale bars, 50 m.

3.7. Ethyl Acetate (EA) Fraction of L. globosus Suppress
the FA-Induced Phosphorylation of c-Jun N-Terminal
Kinase (JNK)
The c-Jun N-terminal kinase (JNK) pathway is important
in modulating cellular responses to inflammation and
oxidative stress. Since FA is an inflammatory mediator, we
estimated the phosphorylation levels of JNK in serum and
brain after 7 day of FA treatment. There was approximately
10-fold significant increase of serum (Fig. 6A) and brain (Fig.
6B) phospho-JNK levels above the baseline. Co-
administration of EA fraction of L. globosus completely
abolished the FA-induced phospho-JNK levels both in liver
and brain, suggesting the potential anti-inflammatory and
anti-oxidant effects of the fraction.

Fig. 6. FA-induced activation of JNK in mouse liver and brain. (A) FA-induced Phosphorylation of JNK in mouse liver tissue. (B) FA-induced Phosphorylation
of JNK in mouse brain tissue. Samples were collected from liver and brain tissues, 7 days after FA induction and were lysed, separated in SDS-PAGE, blotted
and probed with anti-phospho-JNK, anti-JNK and anti- actin. (n=3).
91 Alpana Khatun et al.: Molecular Mechanism of Formalin-Induced Toxicity and Its Management
4. Discussion
The serum enzyme levels are direct measure of hepatic
injury and they show the status of the liver. The elevation of
enzymes induced by FA causes hepatotoxicity which may be
due to its metabolite, a free radical that binds to lipoprotein
and leads to peroxidation of lipids of endoplasmic reticulum.
The disturbance in the transport function of the hepatocytes
as a result of hepatic injury causes the leakage of enzymes
from cells due to altered permeability of membrane
(Zimmerman and Seeff 1970). The increased serum GOT and
GPT level is evidence that these enzymes play an important
role in the development of acute and chronic inflammation
(Anderson, Bocklehurst et al. 1971).
In this study, we found that EA fractions of L. globosus
significantly suppressed the FA-induced elevated SGOT and
SGPT levels (Fig. 3). Most of the anti-inflammatory drugs
exert their beneficial effect by inhibiting either release of
these enzymes or by stabilizing lysosomal membrane which
is one of the major events responsible for the inflammatory
process (Nair, Ravishankar et al. 1988). Thus it can be
assumed that EA fractions of L. globosus might be acting by
either inhibiting the enzymes or stabilizing the membrane.
The elevated level of MDA, which may be due to the free
radicals, is responsible for damaging cell membranes and
further intensifies inflammatory damage (Telang, Chatterjee
et al. 1990). The inflammatory tissue damages could be due
to the liberation of reactive oxygen species from phagocytes
that invades the inflammatory sites (Conner and Grisham
1996).
In the present study, we found that the concentration of
MDA in brain and liver tissues was found to be higher in FA-
induced mice, which were reduced significantly by treatment
with the EA fractions of L. globosus (Fig. 4). The increased
MDA levels in the serum and brain also comprise with the
previous report by Teng et al. (Teng, Beard et al. 2001).
We demonstrated that EA fractions of methanolic extracts
of L. globosus exhibited potential antioxidant and free radical
scavenging capacity (Fig. 1) due to the presence of
flavonoids and phenolic compounds (Table 1). Hence, we
assumed that the protective effect of EA fraction could be the
result of direct free radical scavenging properties (Gurel,
Coskun et al. 2005) or by reacting with membrane
phospholipid bilayers to break the chain reaction initiated by
ROS (Verma and Nair 2001). Histological analyses also
supported the protective effects of EA fractions of L.
globosus (Fig. 5).
Finally, we discovered the molecular mechanism of the
ameliorative effects of EA fraction of L. globosus against
organ toxicity by investigating the intracellular signaling
events. Our results clearly shows that FA induction caused
the activation of oxidative stress responsive JNK pathway, a
member of the mitogen-activated protein kinase (MAPK)
family in the brain and liver, which were completely
suppressed by the supplementation of EA fractions of L.
globosus. All isoforms of JNK are constitutively expressed in
the liver and brain, which plays a key role in cell death and
hepatotoxicity. JNK activation has been well recognized in
both rodent and human liver diseases (Malhi, Bronk et al.
2006, Puri, Mirshahi et al. 2008, Wang, Ausman et al. 2008).
5. Conclusion
FA induction caused the elevation of liver biomarkers;
SGOT and SGPT and lipid peroxidation biomarker, MDA in
liver and brain tissues, which were suppressed towards
normal levels by the supplementation of the EA fractions of
L. globosus presumably via the suppression of oxidative
stress responsive JNK pathway by its antioxidant and free
radical scavenging activity. Although more detailed
mechanisms need to be further investigated, the present
work provides a potential strategy for treating liver and
brain damage. Therefore, the modulation of JNK pathway
could be a good target in the treatment of liver and brain
toxicity.
Acknowledgements
This work was supported in part by the Ministry of
Education, Peoples Republic of Bangladesh.
Abbreviations
FA, formalin; SGOT, serum glutamate oxalate
transaminase; SGPT, serum glutamate pyruvate transaminase;
MDA, malondialdehyde; EA, ethyl acetate; ME, methanol
extract; PE, petroleum ether; CF, chloroform; MAPK,
mitogen activated protein kinase; DPPH, , -diphenyl--
picrylhydrazyl.
References
[1] Anderson, A. J., et al. (1971). "Evidence for the role of
lysosomes in the formation of prostaglandins during
carrageenan induced inflammation in rat." Pharmacol Res
Comm 3: 13-17.
[2] Bakand, S., et al. (2005). " In vitro cytotoxicity testing of
airborne formaldehyde collected in serum-free culture media."
Toxicol Ind Health 21(7-8): 147-154.
[3] BC, V. W., et al. (1993) "Ulosantoin, a potent insecticide from
the sponge Ulosa ruetzleri." J Org Chem 58: 335-337
[4] Berlett, B. S. and E. R. Stadtman (1997). "Protein oxidation in
aging, disease, and oxidative stress." J Biol Chem 272(33):
20313-20316.
[5] Conner, E. M. and M. B. Grisham (1996). "Inflammation, free
radicals, and antioxidants." Nutrition 12(4): 274-277.
[6] Draper, H. and M. Hadley (1990). "Malondialdehyde
determination as index of lipid peroxidation." Methods
Enzymol 186: 421-431.
 American Journal of Life Sciences 2015; 3(2): 85-92
[7] Flyvholm, M. A. and P. Andersen (1993). " Identification of
formaldehyde releasers and occurrence of formaldehyde and
formaldehyde releasers in registered chemical products." Am J
Ind Med 24(5): 533-552.
[8] Gurel, A., et al. (2005). "Vitamin E against oxidative damage
caused by formaldehyde in frontal cortex and hippocampus:
biochemical and histological studies." J Chem Neuroanat 29:
173-178.
[9] Heck, H. D., et al. (1990). "Formaldehyde toxicitynew
understanding." Crit Rev Toxicol 20(6): 397-426.
[10] Khan, M. R. I., et al. (2010). "Antidiabetic Effects of the
Different Fractions of Ethanolic Extracts of Ocimum sanctum
in Normal and Alloxan Induced Diabetic Rats." J Sci Res 2(1):
158-168.
[11] Kilburn, K. H. (1994). "Neurobehavioral impairment and
seizures from formaldehyde." Arch Environ Health 49(1): 37-
44.
[12] Kumar, K. S., et al. (2007). "Antioxidant potential of solvent
extracts of Kappaphycus alvarezii (Doty) Doty An edible
seaweed." Food Chemistry 107: 289-295.
[13] Malhi, H., et al. (2006). "Free fatty acids induce JNK-
dependent hepatocyte lipoapoptosis." J Biol Chem. 281:
1209312101.
[14] Nair, R. B., et al. (1988). "Anti inflammatory effect of
Strbilanthus heyneanusLeaves-A biochemical study." J Res Ay
Sid 9(1-2): 46.
[15] Pan, Y., et al. (2008). "Antioxidant activity of microwave-
assisted extract of longan (Dimocarpus Longan Lour.) peel."
Food Chemistry 106(3): 1264-1270.
[16] Prieto, P., et al. (1999). "Spectrophotometric quantitation of
antioxidant capacity through the formation of a
Phosphomolybdenum Complex: Specific application to the
determination of vitamin E." Analytical Biochemistry 269:
337-341.
[17] Puri, P., et al. (2008). "Activation and Dysregulation of the
Unfolded Protein Response in nonalcoholic fatty liver disease.
Gastroenterology 134: 568-576.
[18] Riley, P. A. (1994). "Free radicals in biology: oxidative stress
and the effects of ionizing radiation." Int J Radiat Biol 65(1):
27-33.
92
[19] Saito, Y., et al. (2005). " Cytotoxic effect of formaldehyde
with free radicals via increment of cellular reactive oxygen
species." Toxicology 210(2-3): 235-245.
[20] Sakanashi, T. M., et al. (1996). "Influence of maternal folate
status on the developmental toxicity of methanol in the CD-1
mouse." Teratology 54(4): 198-206.
[21] Telang, R. S., et al. (1990). "Study on analgesic and
antiinflammatory avtivities of Vitex negunda Linn." Gen
Pharmacol 31(5): 363-366.
[22] Teng, S., et al. (2001). "The formaldehyde metabolic
detoxification enzyme systems and molecular cytotoxic
mechanism in isolated rat hepatocytes." Chem Biol Interact
130-132(1-3): 285-296.
[23] Teng, S., et al. (2001). "The formaldehyde metabolic
detoxification enzyme systems and molecular cytotoxic
mechanism in isolated rat hepatocytes." Chem Biol Interact
130-132(1-3): 285-296.
[24] Thannickal, V. J. and B. L. Fanburg (2000). "Reactive oxygen
species in cell signaling." Am J Physiol Lung Cell Mol
Physiol 279(6): 1005-1008.
[25] Verma, R. J. and A. Nair (2001). "Amerliorative effect of
Vitamin E on aflatoxin-induced lipid peroxidation in the testis
of mice." Asian J Androl 3: 217-221.
[26] Wang, Y., et al. (2008). "Increased apoptosis in high-fat diet-
induced nonalcoholic steatohepatitis in rats is associated with
c-Jun NH2-terminal kinase activation and elevated
proapoptotic Bax." J Nutr 138: 1866-1871.
[27] Wiseman, H. and B. Halliwell (1996). " Damage to DNA by
reactive oxygen and nitrogen species: role in inflammatory
disease and progression to cancer." Biochem J 313: 17-29.
[28] Yu, P. H., et al. (2003). "Physiological and pathological
implications of semicarbazide-sensitive amine oxidase."
Biochim Biophys Acta 1647(1-2): 193-199.
[29] Zhishen, J., et al. (1999). "The determination of flavonoid
contents in mulberry and their scavenging effects on
superoxide radicals." Food Chemisty 64(4): 555-559.
[30] Zimmerman, H. J. and L. B. Seeff (1970). "Enzymes in
hepatic disease." Diagnostic Enzymology. 1, Lea and Febiger,
Philadelphia, USA.